The long-range goal of this project is to investigate the mechanism of action of the peptide hormone glucagon and to contribute to the understanding of its role in the pathophysiology of diabetes mellitus. The principal approach towards this goal is to design and synthesize analogues of glucagon that will bind with high affinity to the glucagon receptor but will not activate adenylyl cyclase. These peptide analogues are expected to provide insight into the structural basis of glucagon action at the molecular level and should be potent antagonists of the hormone. A second complementary approach is to investigate structure-function relationships in the glucagon receptor by site-directed mutagenesis and the biochemical and pharmacological characterization of mutant receptors. Specifically, we will test the two-site binding model of the receptor and determine the residues of the receptor that dictate ligand selectivity. To augment both approaches, structural and biophysical methods will be used. We will determine the structure of glucagon bound to the Nterminal domain of the receptor and to the intact receptor by nuclear magnetic resonance (NMR) analysis. The dynamics of glucagon interaction with receptor and fragments of the receptor will be studied by circular dichroism and fluorescence spectroscopy. An interdisciplinary approach combining chemical synthesis, molecular biology, and structural and biophysical methods is very likely to advance the understanding of glucagon-mediated signal transduction and is crucial for the conception of three-dimensional receptor models to be used in the rational design of glucagon antagonists for the management of diabetes.